Conformation of apolipoprotein B-100 in the low density lipoproteins of tangier disease. Identification of localized conformational response to triglyceride content.

The low density lipoproteins (LDL) from patients with Tangier disease are enriched in triglycerides, 27% of LDL mass versus 7% for normal LDL. To study whether this unique LDL core lipid composition affects the surface disposition of apolipoprotein (apo) B-100, we analyzed the LDL by protease digestion and in competitive radioimmunoassays. Limited proteolytic digestion of Tangier LDL by Staphylococcus aureus V8 protease generated a prominent fragment of 120 kDa (cleavage site at residue 1076), which was not visible in similarly digested normal LDL. In competitive radioimmunoassay, Tangier LDL bound weakly to the apoB-specific monoclonal antibody MB20, compared with control LDL. We localized the MB20 epitope between residues 1031 and 1084 of apoB-100, probably very near residue 1076. DNA sequencing of exon 21 of apoB genomic clones (coding for residues 1014-1084) from a Tangier patient revealed no difference from the normal DNA sequence, thus eliminating a protein polymorphism as a basis for the altered protease sensitivity and antibody binding. When the triglyceride contents of Tangier LDL were reduced to 10% of mass by incubation with normal high density lipoproteins, production of the 120-kDa fragment by proteolysis decreased and MB20 binding increased in affinity, implying a change toward normal conformation of apoB-100. Thus, using two independent techniques, proteolytic digestion and binding of monoclonal antibodies, we have demonstrated an alternative conformation of apoB-100 in the vicinity of residue 1076, which reflects the content of triglycerides in the LDL particle.     

Differentiation and retrodifferentiation of U937 cells: reversible induction and suppression of intermediate filament protein synthesis

Significant morphological and functional changes were observed when human monoblastoid U937 tumor cells growing in suspension were induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h to differentiate along the monocyte/macrophage pathway. These include adherence of the cells to each other and to the substratum, alterations in cell-surface antigen expression and cessation of autonomous proliferation. In this study, we show by both, hybridization analysis of RNA and immunoblotting that an enhanced expression of the intermediate filament (IF) subunit proteins vimentin, lamin A and lamin C accompanied the TPA-induced differentiation process. After long-term culture of differentiated U937 cells in the absence of TPA (more than 28 days), however, the adherent cells retracted their pseudopodia, detached and started again to proliferate. This "retrodifferentiation" process, not previously described was paralleled by a rapid down-regulation of both, IF mRNA and protein synthesis back to the level of undifferentiated U937 control cells. These data suggest a functional relationship between the expression of vimentin and lamins A and C and the differentiation process taking place in these cells.     

Differential effect of dexamethasone on the regulation of phospholipase A2 and prostanoid synthesis in undifferentiated and phorbolester-differentiated U937 cells

The human undifferentiated histiocytic cell-line U937 can be induced to differentiate by incubation with 12-0-tetradecanoylphorbol-13-acetate (TPA) into macrophage-like cells. Dexamethasone reduced the prostaglandin production in TPA-differentiated U937 cells dose dependently, whereas undifferentiated U937 cells were dexamethasone insensitive. Concomitantly phospholipase A2, the enzyme liberating the prostaglandin precursor arachidonic acid, was inhibited by dexamethasone in TPA-differentiated but not in undifferentiated U937 cells. The activity of lysophosphatide acyltransferase, the key enzyme of fatty acid reacylation into phospholipids, remained unchanged both in undifferentiated and TPA-differentiated U937 cells. The data suggest that responsiveness to glucocorticoid-dependent regulation of prostanoid synthesis is acquired by cells of the monocyte-macrophage lineage late in differentiation.     

A reassessment of the phosphoglycerate bypass enzymes in human erythrocytes.

Dissociation of the human erythrocyte into cytoplasmic and membranous components, shows that all of the cell's intrinsic 2,3-diphosphoglycerate phosphatase activity is associated with the soluble component. Further fractionaction of the cytoplasm on DEAE cellulose illustrates that both 1,3-diphosphoglycerate mutase and 2,3-diphosphoglycerate phosphatase activities occur coincidently within one peak. Thermal denaturation of the peak proteins at 60° results in a parallel loss in phosphatase and mutase activity. The identical phenomenon is observed in the presence of the 2,3-diphosphoglycerate phosphatase activator, 2-phosphoglycolate. Homogeneous 1,3-diphosphoglycerate mutase, which quantitatively accounts for all of the intrinsic 2,3-diphosphoglycerate phosphatase within the red cell, also exhibits thermal instability at 60°. These findings suggest that the phosphoglycerate bypass in erythrocytes is under the control of a single, bifunctional enzyme.     

Characterization of recombinant human factor VIII

Recently, complete human factor VIII DNA clones have been obtained and subsequently expressed in baby hamster kidney cells (Wood, W. I., Capon, D. J., Simonsen, C. C., Eaton, D. L., Gitschier, J., Keyt, B., Seeburg, P. H., Smith, D. H., Hollingshead, P., Wion, K. L., Delwart, E., Tuddenham, E. G. D., Vehar, G. A., and Lawn, R. M. (1984) Nature 312, 330-337). The recombinant factor VIII (rVIII) protein secreted from these cells has now been purified allowing its structural analysis and comparison to plasma-derived factor VIII (pdVIII). Analysis of purified rVIII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it consists of multiple polypeptides with relative mobilities (Mr) ranging from 80,000-210,000. The same pattern of polypeptides is also observed for pdVIII resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins associated with rVIII are recognized by pdVIII antibodies in a Western blot. When rVIII and pdVIII are subjected to isoelectric focusing they are resolved into a similar pattern of protein bands. Thrombin, factor Xa, and activated protein C, which modulate factor VIII activity by proteolysis, process rVIII in the same manner they do pdVIII. As is the case for pdVIII, thrombin activation of rVIII coagulant activity correlates with the generation of subunits with Mr of 73,000, 50,000 and 43,000. These subunits appear to form a metal-(perhaps Ca2+) linked complex. EDTA inactivates thrombin-activated rVIII and pdVIII, with the activity being regenerated after the addition of a molar excess of MnCl2. The results suggest that rVIII is structurally and functionally very similar to pdVIII.     

Binary mixtures containing isomers of nitrobenzo[a]pyrene induce greater-than-additive mutational responses in Salmonella typhimurium. I. Analysis by the total concentration-proportional mixture model

The inhibition of cell division induced by bleomycin (BM) and UV irradiation in the set of rec mutants of E. coli K12 was studied. Data presented in this work indicate that BM treatment requires mainly the RecBC pathway for the induction of cell filamentation. In the recB21 mutant cell filamentation is delayed and reduced compared to the wild type. Cell filamentation is BM-induced with similar kinetics in strains with a proficient RecBC recombination pathway (rec+, recF143 and recN262), as well as in the strain with a fully expressed RecF pathway (recB21recC22sbcB15). Induction is completely abolished in the recB21recF143 double mutant. On the other hand cell filamentation was induced similarly by UV irradiation in all strains with a functional recF gene and in the strain with a fully operative RecF pathway, but it was delayed in the recF143 and recB21recF143 mutants.     

Nippostrongylus brasiliensis: effect of dexamethasone upon anthelmintic efficacy

A study has been made to determine the chemotherapeutic susceptibility of natural and “steroid sustained” Nippostrongylus brasiliensis infections in rats. Dexamethasone in the drinking water (2.5 × 10^?4 mg/ml) aided establishment of greater parasite populations but restricted growth of young rats. Comparison trials with thiabendazole, cambendazole, and parbendazole, as well as 10 candidate drugs, showed that the parasiticidal activity of these materials was not significantly altered between the 10-day-old naturally maintained and the 17-day-old steroid-treated parasitic infections. As expected, 17-day-old naturally maintained parasite populations were highly susceptible to the anthelmintic action of these materials. In one instance, the toxic response elicited by a candidate drug was apparently potentiated by dexamethasone.     

A spontaneous outbreak of polycholorinated biphenyl (PCB) toxicity in rhesus monkeys (Macaca mulatta): toxicopathology.

Polychlorinated biphenyls (PCBs) were shown to be the cause of a wasting syndrome and reproductive dysfunction in a group of rhesus monkeys (Macaca mulatta). The polychlorinated biphenyls were found by gas chromatographic analysis in high amounts in the superficial layers of concrete slab floors in the housing facility. The concrete sealant was suspected as the original source although it is not known whether it contained polychlorinated biphenyls before it was applied or if it was contaminated later. Histopathologic findings for the most part were confined to epithelial tissues where squamous metaplasia of several glandular tissues was observed. A striking finding was severe hypertrophy of the glandular stomach and a similar but less severe lesion in the colon. The lesions may be related in part to impaired vitamin A metabolism, but not to a dietary deficiency.